Cell culture device

ABSTRACT

The invention relates to a device that is intended, in particular, for the culture of cells. The inventive device consists of a chamber ( 100 ) which is intended to receive the cells ( 10 ) and which comprises at least one liquid injection conduit ( 121, 122, 123, 124, 125, 126 ), the surface of the liquid contained in said chamber ( 100 ) being in free contact with the air. The invention is characterised in that it comprises a collector element ( 40 ) which can form at least one liquid bridge with the free surface of the liquid, said collector element ( 40 ) comprising a mouth piece ( 43 ) which is positioned essentially at the level of the free surface of the liquid contained in the chamber ( 100 ). The aforementioned mouth piece ( 43 ) is maintained at a vacuum such that it absorbs a flow of air which moves the surface film ( 30 ) of the liquid via the liquid bridge.

The present invention relates to a cell culture device which allows thetreatment thereof with various media while avoiding the handlingthereof.

Document FR 2 659 347 (published on Sep. 13, 1991) describes a cellculture device comprising a chamber intended to receive the cells. Thechamber comprises a horizontal support formed by two glass plates thatare juxtaposed and spaced apart so as to form between them a slot havinga width which is less than the diameters of the cells. The cells to betreated are placed on the slot. This chamber is intended to containliquid culture or pulse media. The various liquid media are injectedsuccessively into the chamber via distinct tubes placed above the wallsupporting the cells. Part of the liquid contained that is injected intothe chamber is evacuated by one or more tube(s) located below the wall.Another part of this liquid is evacuated by overflow.

In this device, the cells are held on the slot by virtue of the reducedpressure caused by the suction of the liquid medium by suction via theevacuation tube(s).

Such a device is intended in particular for culturing ovocytes,fertilized eggs or embryos, etc.

In particular, it can be used for activating experimental ovocytes, thisactivation being necessary for the correct subsequent development of theembryos. In order to cause this activation, a culture medium is injectedinto the chamber and the cells to be activated are placed in thisculture medium. The ion-rich culture medium is then evacuated andsimultaneously replaced with a pulse medium containing Ca²⁺ ions. Whenthe culture medium has been completely evacuated and replaced with thepulse medium, the cells are subjected to a pulse from an electric field,which causes the transient electropermeabilization of their plasmamembrane and the penetration of the Ca²⁺ ions into the cells. The pulsemedium is then, in turn, evacuated and replaced with the culture medium.These steps are repeated a certain number of times such that the cellsare subjected to a controlled series of calcium pulses which triggerstheir activation.

An advantage of this device is that it makes it possible to treat thecells with various media while avoiding the handling of the cells.

However, a problem posed by this type of device is that the replacing ofone medium with another is relatively long, which limits the frequencyof alternation of the media in the chamber.

For example, when the device is used for activating cells, a minimumtime for injecting the pulse medium is necessary (of the order of 40seconds) for replacing the culture medium with the pulse medium. Thisminimum time in fact guarantees sufficient washing of the cells by thepulse medium.

Should this minimum washing time not be observed, the pulse medium wouldcontain residual ions originating from the culture medium. When theelectric field was applied, these ions would induce a transmembrane ioncurrent which could cause the cells to be destroyed.

Another problem related to the washing is that the prolonged exposure ofthe cells in the pulse medium having a low ionic strength disturbs theequilibrium of the cells and exposes them to deleterious effects. Inorder to preserve the cell survival, it is therefore necessary to reducethe washing time of the cells.

One aim of the invention is to provide a cell culture device which makesit possible to rapidly replace the medium in which the cells are placed.

It has been noted that evacuation of the liquid by overflow is anon-uniform phenomenon. In fact, during the injection of the liquid intothe chamber, the free surface of the liquid rises above the upper planeof the enclosure such that the surface of the liquid takes the form of aconvex meniscus. This phenomenon is related to the surface tensionswhich are exercised at the surface of the liquid and to the wettabilityof the vertical walls of the enclosure with this liquid. The differencein level of the liquid above the edges of the enclosure must reach acritical value in order for the equilibrium between, firstly, the forcesrelated to the surface tensions and, secondly, the forces related togravity to be broken. This break results in the surplus liquid flowingover the walls of the enclosure.

It results therefrom that the evacuation of the liquid by overflowoccurs in a discontinuous and unpredictable manner. In particular, itmay be that, during a phase of replacement of one medium with another,the medium to be replaced is not evacuated. This evacuation is nottherefore satisfactory since it does not guarantee the washing of thecells during this replacement phase.

Moreover, another problem is that this discontinuous flow generates asuccession of shockwaves in the liquid. These shockwaves move the cellson the support.

The cells have a tendency to move along the slot of the support elementtoward the center of the enclosure, where they come together. They arecompressed against one another. The decrease in space between the cellsmodifies the efficiency of the washing at the periphery of each cell.

Finally, yet another problem is that the interface between the culturemedium and the surrounding gas is the site of surface phenomena. Atomicor molecular films form at the free surface of the liquid. These filmsare not renewed when one medium is replaced with another in the chamber.

When the culture device is used for applying electric field pulses tothe cells, the films can constitute conductivity bridges between theelectrodes. These conductivity bridges establish a short circuit betweenthe electrodes. As a result of this, there is a significant decrease inthe effectiveness of the electric field applied.

To overcome these drawbacks, the invention proposes a device intended inparticular for culturing cells, comprising a chamber intended to receivethe cells, said chamber comprising at least one liquid injectionconduit, the surface of the liquid contained in the chamber being infree contact with the air, characterized in that it comprises acollector element which can form at least one liquid bridge with thefree surface of the liquid, the collector element comprising amouthpiece which is placed substantially at the level of the freesurface of the liquid contained in the chamber, the mouthpiece beingkept at low pressure such that it absorbs a flow of air which moves thesurface film of the liquid via the liquid bridge.

The device of the invention makes it possible to create a liquid bridgein the form of a meniscus between the free surface of the liquid and thecollector element. This meniscus regulates, by means of an equilibriumeffect, the surface tensions at the surface of the liquid contained inthe chamber.

Such a device makes it possible, by virtue of the flow of air, tocontinuously eliminate the surplus liquid in the chamber and thus tomaintain a constant level of liquid.

It also makes it possible to obtain a permanent renewal of the surfacefilm of the liquid of the chamber.

In one embodiment of the invention, the collector element is placed on arim of a wall of the chamber, its mouthpiece being set back from thewall.

Preferably, a portion of the rim located in front of the mouthpiece ofthe collector element is covered with a hydrophilic substance.

Preferably, the mouthpiece is set back from the wall at a distance d ofbetween 10 and 30 μm.

Preferably, the mouthpiece of the collector is elongated in shape andextends longitudinally along the edge of the wall.

In one embodiment of the invention, the opening is of the order of 2 to4 mm in length.

In one embodiment of the invention, the opening is of the order of 0.15to 0.30 mm in height.

In one embodiment of the invention, the mouthpiece of the collectorelement has a generally rectangular shape.

Advantageously, the device comprises several elements placed along theedges of the walls of the chamber.

The invention also relates to a method for regulating the level of aliquid contained in a chamber intended to receive cells, said chambercomprising at least one liquid injection conduit, the surface of theliquid contained in the chamber being in free contact with the air,characterized in that at least one liquid bridge is formed with the freesurface of the liquid and a flow of air is drawn by suction in thevicinity of the liquid bridge, such that this flow of air moves thesurface film of the liquid via the liquid bridge.

Other characteristics and advantages will further emerge from thefollowing description, which is given purely by way of non-limitingillustration and should be read with regard to the attached figures,among which:

FIG. 1 is a representative diagram of an example of a cell culturedevice in accordance with an embodiment of the invention,

FIG. 2 is a detailed diagram of a collector of the device in FIG. 1,

FIG. 3 is a sectional diagram of the collector in FIG. 2.

In FIG. 1, the cell culture device represented comprises a chamber 100comprising walls 11, 12 and 13 delimiting an enclosure 20 intended tocontain a liquid medium. The enclosure is filled with a liquid mediumcorresponding to the ongoing cell treatment phase. In enclosure 20, ahorizontal support element is positioned, formed by the juxtaposition oftwo glass plates 101 and 102. The glass plates 101 and 102 are heldembedded in the side walls 11 and 12 of the chamber. These glass plates101 and 102 are set apart so as to define between them a rectilinearslot 103 having a width that is less than the diameter of the ovocytes10 intended to be treated.

The chamber 100 also comprises electrodes 111 and 112 which extendlongitudinally on either side of the slot 103.

The culture liquid is brought to the upper part of the enclosure 20 by aseries of conduits, the mouthpieces 121, 122, 123, 124, 125 and 126 ofwhich are placed at regular intervals along the walls 11, 12 and 13.Moreover, a part of the liquid contained in the chamber is evacuated bymeans of an evacuation conduit 104 located at a level lower than that ofthe support element. The stream of liquid keeps the ovocytes 10 stuck tothe slot 103 by means of low pressure.

The free surface of the liquid contained in the chamber 100 forms amolecular film 30 which is convex in shape. This film 30 consists of analignment of oriented molecules, the hydrophilic function of whichpositions itself toward the fluid and the hydrophobic portion of whichpositions itself toward the outer air.

In order to eliminate the molecular film 30 and to absorb the surplusliquid contained in the enclosure 20, the culture device comprisescollectors 40 placed on a horizontal rim 21 of the wall 11 of theenclosure 20.

In a symmetrical manner, the device may comprise collectors placed onrims 22 or 23 of the walls 12 or 13 (at the positions represented bydashed lines).

These collectors 40 may be placed at regular intervals around theenclosure 20.

FIG. 2 is a diagram representing in greater detail a collector 40 of thecell culture device in FIG. 1.

This collector 40 is placed on the rim 21 of the wall 11 of the chamber,set back from this wall by a distance d. It comprises a hollow body 41which has a rectangular opening 42 extending along the wall 11. Thisopening has a length of L=2 mm and a height of l=0.2 mm.

Two liquid bridges 51 and 52 are formed between the surface 30 of theliquid contained in the enclosure 20 and the vertical edges of theopening 42. These liquid bridges 51 and 52 are formed by distortions ofthe surface 30 of the liquid (or menisci) in contact with the edges ofthe opening 42.

The collector 40 comprises, moreover, an evacuation conduit 43 connectedto a suction device, which is not represented. This suction device makesit possible to draw by suction the air contained in the collector 40.This suction causes air to circulate through the opening 42, which movesan upper portion of the liquid contained in the chamber via the liquidbridges 51 and 52. As a result of this, the molecular film 30 at thesurface of the liquid is permanently moved by the air circulating in thecollector and eliminated from the surface of the liquid.

The length L of the opening was chosen so as to be at least twice thecapillary length k⁻¹ of the liquid contained in the chamber. Thischaracteristic guarantees the formation of two lateral menisci andtherefore the non-sealing of the opening 42 by the liquid. The air istherefore always free to circulate through the opening 42.

The liquids generally used for treating the cells generally havecapillary lengths of between 1 mm and 2 mm. The length L of the openingwill therefore preferably be of the order of 2 to 4 mm.

The collector 40 is set back from the wall by a distance d. In fact, theopening should not be too close to the wall 11, since, in this case, thecapillarity forces would result in the formation of a single liquidbridge which would seal the opening 42 and which would empty out of theenclosure 20 the liquid that it contains.

However, the opening 42 should not be too far from the wall 11, since,in this case, the collector would no longer have any effect on theliquid.

Moreover, a distance d of a few microns results in the formation of aconcave meniscus. Now, the concave shape has several drawbacks:

-   -   firstly, it increases surface tensions toward the bottom of the        enclosure 20, in the vicinity of the cells 10, which makes it        more delicate for the operator to position or remove the cells,    -   secondly, the concave shape modifies the optical path of the        light and makes it more difficult for the operator to see the        cells.

Consequently, it is desirable to maintain a meniscus with a stableconvex shape. For this reason, the distance d is preferably between 10and 30 μm.

Thus, in the collector 40 represented in FIG. 2, the distance d is 20μm.

Preferably, the rims 21, 22 and 23 of the walls 11, 12 and 13 of thechamber are covered with a hydrophobic substance, except in front of theopenings 42 of the collectors, where they are covered with a hydrophilicsubstance. This arrangement promotes the initiation of a liquid bridgeat the level of the collectors 40.

In FIG. 3, a collector 40 is shown in the form of a section along theevacuation conduit 43. The path of the air drawn by suction isrepresented by arrows.

1. A device intended in particular for culturing cells, comprising a chamber (100) intended to receive the cells (10), said chamber (100) comprising at least one liquid injection conduit (121, 122, 123, 124, 125, 126), the surface of the liquid contained in the chamber (100) being in free contact with the air, characterized in that it comprises a collector element (40) which can form at least one liquid bridge (51, 52) with the free surface of the liquid, the collector element (40) comprising a mouthpiece (42) which is placed substantially at the level of the free surface of the liquid contained in the chamber (100), the mouthpiece (42) being kept at low pressure such that it absorbs a flow of air which moves the surface film (30) of the liquid via the liquid bridge (51, 52).
 2. The device as claimed in claim 1, characterized in that the collector element (40) is placed on a rim (21) of a wall (11) of the chamber (100), its mouthpiece (42) being set back from the wall (11).
 3. The device as claimed in claim 2, characterized in that a portion of the rim (21) located in front of the mouthpiece (42) of the collector element (40) is covered with a hydrophilic substance.
 4. The device as claimed in either of claims 2 and 3, characterized in that the mouthpiece is set back from the wall (11) at a distance (d) of between 10 and 30 μm.
 5. The device as claimed in any one of the preceding claims, characterized in that mouthpiece (42) of the collector element (40) is elongated in shape and extends longitudinally along the edge of the wall (11).
 6. The device as claimed in claim 5, characterized in that the opening (42) is of the order of 2 to 4 mm in length.
 7. The device as claimed in either of claims 5 and 6, characterized in that the opening (42) is of the order of 0.15 to 0.30 mm in height.
 8. The device as claimed in one of the preceding claims, characterized in that the mouthpiece (42) has a generally rectangular shape.
 9. The device as claimed in one of the preceding claims, characterized in that it comprises several collector elements (40) placed along the edges of the walls (11, 12, 13) of the chamber (100).
 10. A method for regulating the level of a liquid contained in a chamber (100) intended to receive cells (10), said chamber (100) comprising at least one liquid injection conduit (121, 122, 123, 124, 125, 126), the surface of the liquid contained in the chamber being in free contact with the air, characterized in that at least one liquid bridge (51, 52) is formed with the free surface of the liquid and a flow of air is drawn by suction in the vicinity of the liquid bridge (51, 52), such that this flow of air moves the surface film (30) of the liquid via the liquid bridge (51, 52). 